Ozone treatment of biomass to enhance enzymatic saccharification

ABSTRACT

Methods for treating lignocellulosic biomass to produce readily saccharifiable carbohydrate-enriched biomass are provided. In one method, lignocellulosic biomass comprising lignin is treated with aqueous ammonia, then contacted with a gas comprising ozone at a temperature of about 0° C. to about 50° C. In another method, lignocellulosic biomass comprising lignin is contacted with a gas comprising ozone at a temperature of about 0° C. to about 50° C., then treated with aqueous ammonia. The readily saccharifiable carbohydrate-enriched biomass may be saccharified with an enzyme consortium to produce fermentable sugars.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims benefit of priority from Provisional ApplicationNo. 61/139,116 filed Dec. 19, 2008. This application hereby incorporatesby reference Provisional Application No. 61/139,116 in its entirety.

FIELD OF THE INVENTION

Methods for producing readily saccharifiable, carbohydrate-enrichedlignocellulosic biomass are provided and disclosed. Specifically,pretreated biomass may be prepared by treating under conditions of highsolids and low ammonia concentration, then by contacting with a gascomprising ozone. The remaining carbohydrate-enriched solids in thepretreated biomass may then be subjected to enzymatic saccharificationto obtain fermentable sugars, which may be subjected to furtherprocessing for the production of other target products.

BACKGROUND OF THE INVENTION

Cellulosic and lignocellulosic feedstocks and wastes, such asagricultural residues, wood, forestry wastes, sludge from papermanufacture, and municipal and industrial solid wastes, provide apotentially large renewable feedstock for the production of chemicals,plastics, fuels and feeds. Cellulosic and lignocellulosic feedstocks andwastes, composed of carbohydrate polymers comprising cellulose,hemicellulose, pectins and lignin are generally treated by a variety ofchemical, mechanical and enzymatic means to release primarily hexose andpentose sugars, which can then be fermented to useful products.

Pretreatment methods are usually used to make the polysaccharides oflignocellulosic biomass more readily accessible to cellulolytic enzymes.One of the major impediments to cellulolytic enzyme digest ofpolysaccharide is the presence of lignin, a barrier that limits theaccess of the enzymes to their substrates, and a surface to which theenzymes bind non-productively. Because of the significant cost of enzymein the pretreatment process, it is desirable to minimize the enzymeloading by either inactivation of the lignin to enzyme adsorption or itsoutright extraction. Another challenge is the inaccessibility of thecellulose to enzymatic hydrolysis either because of its protection byhemicellulose and lignin or by its crystallinity. Pretreatment methodsthat attempt to overcome these challenges include: steam explosion, hotwater, dilute acid, ammonia fiber explosion, alkaline hydrolysis(including ammonia recycled percolation), oxidative delignification,organosolv, and ozonation.

Previously applied pretreatments methods often suffer from shortcomings,including separate hexose and pentose streams (e.g. dilute acid),inadequate lignin extraction or lack of separation of extracted ligninfrom polysaccharide, particularly in those feedstocks with high lignincontent (e.g., sugar cane bagasse, softwoods), disposal of wasteproducts (e.g., salts formed upon neutralization of acid or base), andpoor recoveries of carbohydrate due to breakdown or loss in wash steps.Other problems include the high cost of energy, capital equipment, andpretreatment catalyst recovery, and incompatibility withsaccharification enzymes.

Ben-Ghedalia et al. (in J. Sci. Food Agric. 1980, 31(12), 1337-1342)disclose treatment of cotton straw with ammonium hydroxide (at roomtemperature for 60 days), ozone treatment, and combined ammoniumhydroxide treatment followed by ozonation. They report that ozonetreatment caused a 50% reduction in lignin and hemicellulose, and acorresponding increase in cell contents. In vitro organic matterdigestibility, as measured by the rumen liquor-acid pepsin method, wasincreased by more than 100% as a result of the partial conversion ofcell walls into cell contents and the increased digestibility of thecell walls. Cellulose in vitro digestibility was increased by thecombined treatment as well. No information on sugar recovery wasprovided.

One of the major challenges of the pretreatment of lignocellulosicbiomass is to maximize the extraction or chemical neutralization (withrespect to non-productive binding of cellulolytic enzymes) of the ligninwhile minimizing the loss of carbohydrate (cellulose plushemicellulose). The higher the selectivity, the higher the overall yieldof monomeric sugars following combined pretreatment and enzymaticsaccharification.

SUMMARY OF THE INVENTION

The present invention provides methods for producing readilysaccharifiable carbohydrate-enriched biomass and for selectivelyoxidizing lignin while retaining carbohydrate in good yield. The methodsinclude treating lignocellulosic biomass under conditions of high solidsand low ammonia concentration, then contacting the biomass with a gascomprising ozone. The methods also include contacting lignocellulosicbiomass with a gas comprising ozone, then treating the biomass underconditions of high solids and low ammonia concentration. With thesemethods, carbohydrate-enriched biomass, highly susceptible to enzymaticsaccharification, is produced in a cost effective process. Followingpretreatment, the carbohydrate-enriched biomass may be further treatedwith a saccharification enzyme consortium to produce high yields offermentable sugars (for example, glucose and xylose). These sugars maybe subjected to further processing, such as bioconversion to value-addedchemicals and fuels.

In one embodiment of the invention, a method is provided, the methodcomprising:

(a) providing lignocellulosic biomass comprising lignin;

(b) contacting the biomass with an aqueous solution comprising ammoniato form a biomass-aqueous ammonia mixture, wherein the ammonia ispresent at a concentration at least sufficient to maintain alkaline pHof the biomass-aqueous ammonia mixture but wherein said ammonia ispresent at less than about 12 weight percent relative to dry weight ofbiomass, and further wherein the dry weight of biomass is at a highsolids concentration of at least about 15 weight percent relative to theweight of the biomass-aqueous ammonia mixture, to produce anammonia-treated biomass; and

(c) contacting the ammonia-treated biomass with a gas comprising ozoneat a temperature of about 0° C. to about 50° C. whereby a readilysaccharifiable carbohydrate-enriched biomass is produced.

In one embodiment of the invention a method is provided, the methodcomprising:

(a) providing lignocellulosic biomass comprising lignin;

(b) contacting the biomass with a gas comprising ozone at a temperatureof about 0° C. to about 50° C.

(c) contacting the ozone-treated biomass with an aqueous solutioncomprising ammonia to form a mixture comprising ozone-treated biomassand aqueous ammonia, wherein the ammonia is present at a concentrationat least sufficient to maintain alkaline pH of the mixture but whereinsaid ammonia is present at less than about 12 weight percent relative todry weight of ozone treated biomass, and further wherein the dry weightof biomass is at a high solids concentration of at least about 15 weightpercent relative to the weight of the mixture, whereby a readilysaccharifiable carbohydrate-enriched biomass is produced.

According to the methods of the invention, the gas comprises about 0.1to about 20 percent by volume ozone. In some embodiments, the gascomprises about 0.5 to about 5 percent by volume ozone. In someembodiments, the gas further comprises air, nitrogen, oxygen, argon, ora combination thereof. In some embodiments, the ratio of ozone toammonia-treated biomass is at least 1:500 on a weight basis. In someembodiments, the ratio of ozone to lignocellulosic biomass is at least1:100 on a weight basis. According to the methods of the invention, insome embodiments the temperature is about 0° C. to about 25° C.

In some embodiments, the methods further comprise applying energy to thelignocellulosic biomass, to the ammonia-treated biomass, or to both. Insome embodiments, the methods further comprise applying energy to thelignocellulosic biomass, to the ozone-treated biomass, or to both. Theapplying energy is by milling, crushing, grinding, shredding, chopping,disc refining, ultrasound, microwave, or a combination of these. In someembodiments, the lignocellulosic biomass, the ammonia-treated biomass,or both contains at least about 30 percent moisture.

In some embodiments, the methods of the invention further comprisesaccharifying the biomass with an enzyme consortium whereby fermentablesugars are produced. In some embodiments, the methods of the inventionfurther comprise fermenting the sugars to produce a target product. Insome embodiments, the target product is selected from the groupconsisting of ethanol, butanol, and 1,3-propanediol.

In some embodiments, the pH of the biomass-aqueous ammonia mixture isgreater than about 8. In some embodiments, the ammonia is present atless than about 10 weight percent relative to dry weight of biomass. Insome embodiments, ammonia is selected from the group consisting ofammonia gas, ammonium hydroxide, urea, and combinations thereof. In someembodiments, the aqueous solution comprising ammonia further comprisesat least one additional base selected from the group consisting ofsodium hydroxide, sodium carbonate, potassium hydroxide, potassiumcarbonate, calcium hydroxide, and calcium carbonate.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for the treatment of biomass inorder to enhance the subsequent enzymatic saccharification step. In onemethod, in the first step biomass at relatively high concentration istreated with a relatively low concentration of ammonia relative to thedry weight of the biomass. Then in the second step, the ammonia-treatedbiomass, as an aqueous suspension or as a solid, is contacted with a gascomprising ozone. The treated biomass may be digested with asaccharification enzyme consortium to produce fermentable sugars. Inanother method, in the first step biomass, as an aqueous suspension oras a solid, is contacted with a gas comprising ozone. Then in the secondstep, the ozone-treated biomass at relatively high concentration istreated with a relatively low concentration of ammonia relative to theweight of the biomass.

Applicants specifically incorporate the entire contents of all citedreferences in this disclosure. Further, when an amount, concentration,or other value or parameter is given as either a range, preferred range,or a list of upper preferable values and lower preferable values, thisis to be understood as specifically disclosing all ranges formed fromany pair of any upper range limit or preferred value and any lower rangelimit or preferred value, regardless of whether ranges are separatelydisclosed. Where a range of numerical values is recited herein, unlessotherwise stated, the range is intended to include the endpointsthereof, and all integers and fractions within the range. It is notintended that the scope of the invention be limited to the specificvalues recited when defining a range.

DEFINITIONS

The following definitions are used in this disclosure:

“Room temperature” and “ambient” when used in reference to temperaturerefer to any temperature from about 15° C. to about 25° C.

“Fermentable sugars” refers to a sugar content primarily comprisingmonosaccharides and some polysaccharides that can be used as a carbonsource by a microorganism in a fermentation process to produce a targetproduct.

“Lignocellulosic” refers to material comprising both lignin andcellulose. Lignocellulosic material may also comprise hemicellulose. Inthe methods described herein, lignin is oxidized and substantiallydegraded to produce a carbohydrate-enriched biomass comprisingfermentable sugars.

“Dissolved lignin” means the lignin that is dissolved in a solvent.

“Al lignin” refers to acid-insoluble lignin.

“Autohydrolysis” refers to the hydrolysis of biomass in the presence ofsolvent (water or organic solvent plus water) plus heat with no furtheradditions, such as without hydrolytic enzymes

“Cellulosic” refers to a composition comprising cellulose.

“Target product” refers to a chemical, fuel, or chemical building blockproduced by fermentation. Product is used in a broad sense and includesmolecules such as proteins, including, for example, peptides, enzymes,and antibodies. Also contemplated within the definition of targetproduct are ethanol and butanol.

The abbreviation “EtOH” refers to ethanol or ethyl alcohol.

“Dry weight of biomass” refers to the weight of the biomass having allor essentially all water removed. Dry weight is typically measuredaccording to American Society for Testing and Materials (ASTM) StandardE1756-01 (Standard Test Method for Determination of Total Solids inBiomass) or Technical Association of the Pulp and Paper Industry, Inc.(TAPPI) Standard T-412 om-02 (Moisture in Pulp, Paper and Paperboard).

“Selective extraction” means removal of lignin while substantiallyretaining carbohydrates.

A “solvent” as used herein is a liquid that dissolves a solid, liquid,or gaseous solute, resulting in a solution.

“Biomass” and “lignocellulosic biomass” as used herein refer to anylignocellulosic material, including cellulosic and hemi-cellulosicmaterial, for example, bioenergy crops, agricultural residues, municipalsolid waste, industrial solid waste, yard waste, wood, forestry waste,and combinations thereof, and as further described below. Biomass has acarbohydrate content that comprises polysaccharides and oligosaccharidesand may also comprise additional components, such as protein and/orlipid.

“Highly conserved” as used herein refers to the carbohydrate content ofthe lignocellulosic material after the processing steps describedherein. In an embodiment of the invention, the highly conservedcarbohydrate content provides for sugar yields after saccharificationthat are substantially similar to theoretical yields and/ordemonstration of minimal loss in sugar yield from the processesdescribed herein. In an embodiment of the invention, highly-conservedwith reference to carbohydrate content refers to the conservation ofgreater than or equal to 85% of the biomass carbohydrate as compared tobiomass prior to pretreating as described herein.

“Preprocessing” as used herein refers to processing of lignocellulosicbiomass prior to pretreatment. Preprocessing is any treatment of biomassthat prepares the biomass for pretreatment, such as mechanicallychopping and/or drying to the appropriate moisture content.

“Aqueous ammonia-treated biomass suspension” refers to a mixture ofammonia-treated biomass and aqueous solution wherein the biomass is insuspension in the aqueous solution. The biomass suspension may compriseadditional components such as a buffer. As used herein, “slurry” is usedinterchangeably with “suspension.”

“Saccharification” refers to the production of fermentable sugars frompolysaccharides by the action of hydrolytic enzymes. Production offermentable sugars from pretreated biomass occurs by enzymaticsaccharification by the action of cellulolytic and hemicellulolyticenzymes.

“Pretreating biomass” or “biomass pretreatment” as used herein refers tosubjecting native or preprocessed biomass to chemical, physical, orbiological action, or any combination thereof, rendering the biomassmore susceptible to enzymatic saccharification or other means ofhydrolysis prior to saccharification. For example, the methods claimedherein may be referred to as pretreatment processes that contribute torendering biomass more accessible to hydrolytic enzymes forsaccharification.

“Pretreated biomass” as used herein refers to native or preprocessedbiomass that has been subjected to chemical, physical, or biologicalaction, or any combination thereof, rendering the biomass moresusceptible to enzymatic saccharification or other means of hydrolysisprior to saccharification.

“Air-drying the filtered biomass” can be performed by allowing thebiomass to dry through equilibration with the air of the ambientatmosphere.

“Readily saccharifiable biomass” means biomass that iscarbohydrate-enriched and made more amenable to hydrolysis bycellulolytic or hemi-cellulolytic enzymes for producing monomeric andoligomeric sugars.

“Carbohydrate-enriched” as used herein refers to the biomass produced bythe process treatments described herein in which lignin in the biomassis selectively oxidized and degraded while biomass carbohydrate isretained in good yield. In one embodiment the readily saccharifiablecarbohydrate-enriched biomass produced by the processes described hereinhas a carbohydrate concentration of greater than or equal to about 85%of the biomass carbohydrate as compared to biomass prior to pretreatingas described herein while removing 75% or greater of the biomass lignin.

“Filtering free liquid under pressure” means removal of unbound liquidthrough filtration, with some pressure difference on opposite faces ofthe filter.

“Air-dried sample” means a pretreated biomass which is allowed to dry atambient temperature to the point where its moisture content isapproximately in equilibrium with that of the ambient air, typically≧85% dry matter.

“Substantially lignin-free biomass” means a pretreated sample containingabout ≦25% of the starting lignin composition.

“Pressure vessel” is a sealed vessel that may be equipped or not with amechanism for agitation of a biomass/solvent suspension, in which apositive pressure is developed upon heating the lignocellulosic biomass.

“Hydrolysate” refers to the liquid in contact with the lignocellulosicbiomass which contains the products of hydrolytic reactions acting uponthe biomass (either enzymatic or not), in this case monomeric andoligomeric sugars.

“Organosolv” means a mixture of organic solvent and water.

“Enzyme consortium” or “saccharification enzyme consortium” is acollection of enzymes, usually secreted by a microorganism, which in thepresent case will typically contain one or more cellulases, xylanases,glycosidases, ligninases and feruloyl esterases.

“Monomeric sugars” or “simple sugars” consist of a single pentose orhexose unit, e.g., glucose.

“Delignification” is the act of removing lignin from lignocellulosicbiomass. In the context of this application delignification meansfragmentation and degradation of lignin from the lignocellulosic biomassusing ozone.

“Fragmentation” is a process in which lignocellulosic biomass is treatedwith ozone to break the lignin down into smaller subunits. In thecontext of the present application, oxidation of the lignin maycontribute to breaking the lignin down into smaller subunits.

An “aqueous solution comprising ammonia” refers to the use of ammoniagas (NH₃), compounds comprising ammonium ions (NH₄ ⁺) such as ammoniumhydroxide or ammonium sulfate, compounds that release ammonia upondegradation such as urea, and combinations thereof in an aqueous medium.

“Ozonation” is the act of treating biomass with ozone. The biomass maybe present in an aqueous suspension or as a solid without an additionalliquid phase.

Methods for pretreating lignocellulosic biomass to produce readilysaccharifiable biomass are provided. These methods provide economicprocesses for rendering components of the lignocellulosic biomass moreaccessible or more amenable to enzymatic saccharification. In thisdisclosure, one pretreatment method involves first treating biomassunder conditions of high solids and low ammonia concentration, thencontacting the biomass with a gas comprising ozone; alternatively, inanother method biomass may first be contacted with a gas comprisingozone, then treated under conditions of high solids and low ammoniaconcentration. The presence of ozone assists lignin fragmentation andcarbohydrate recovery, and a readily saccharifiablecarbohydrate-enriched biomass is produced.

In addition, the methods described in the present disclosure minimizethe loss of carbohydrate during the pretreatment process and maximizethe yield of monomeric sugars in saccharification.

Lignocellulosic Biomass:

The lignocellulosic biomass pretreated herein includes, but is notlimited to, bioenergy crops, agricultural residues, municipal solidwaste, industrial solid waste, sludge from paper manufacture, yardwaste, wood and forestry waste. Examples of biomass include, but are notlimited to, corn cobs, crop residues such as corn husks, corn stover,grasses, wheat, wheat straw, barley, barley straw, hay, rice straw,switchgrass, waste paper, sugar cane bagasse, sorghum, soy, componentsobtained from milling of grains, trees, branches, roots, leaves, woodchips, sawdust, shrubs and bushes, vegetables, fruits, flowers andanimal manure.

In one embodiment, biomass that is useful for the invention includesbiomass that has a relatively high carbohydrate content, is relativelydense, and/or is relatively easy to collect, transport, store and/orhandle.

In one embodiment of the invention, biomass that is useful includes corncobs, corn stover, sugar cane bagasse and switchgrass.

In another embodiment, the lignocellulosic biomass includes agriculturalresidues such as corn stover, wheat straw, barley straw, oat straw, ricestraw, canola straw, and soybean stover; grasses such as switch grass,miscanthus, cord grass, and reed canary grass; fiber process residuessuch as corn fiber, beet pulp, pulp mill fines and rejects and sugarcane bagasse; sorghum; forestry wastes such as aspen wood, otherhardwoods, softwood and sawdust; and post-consumer waste paper products;as well as other crops or sufficiently abundant lignocellulosicmaterial.

The lignocellulosic biomass may be derived from a single source, orbiomass may comprise a mixture derived from more than one source; forexample, biomass could comprise a mixture of corn cobs and corn stover,or a mixture of stems or stalks and leaves.

The biomass may be used directly as obtained from the source, or may besubjected to some preprocessing, for example, energy may be applied tothe biomass to reduce the size, increase the exposed surface area,and/or increase the accessibility of lignin and of cellulose,hemicellulose, and/or oligosaccharides present in the biomass to theaqueous ammonia pretreatment, the ozonation pretreatment, and tosaccharification enzymes. Energy means useful for reducing the size,increasing the exposed surface area, and/or increasing the accessibilityof the lignin, and the cellulose, hemicellulose, and/or oligosaccharidespresent in the biomass to the aqueous ammonia pretreatment, theozonation pretreatment, and to saccharification enzymes include, but arenot limited to, milling, crushing, grinding, shredding, chopping, discrefining, ultrasound, and microwave. This application of energy mayoccur before or during either or both of the pretreatment steps, beforeor during saccharification, or any combination thereof.

Drying biomass prior to pretreatment may occur as well by conventionalmeans, such as by using rotary dryers, flash dryers, or superheatedsteam dryers.

Ammonia Treatment:

The concentration of ammonia used in the present pretreatment methods isminimally a concentration that is sufficient to maintain the pH of thebiomass-aqueous ammonia mixture alkaline and maximally less than about12 weight percent relative to dry weight of biomass. This lowconcentration of ammonia is sufficient for pretreatment, and the lowconcentration may also be less than about 10 weight percent relative todry weight of biomass. A very low concentration of 6 percent ammoniarelative to dry eight of biomass, or less, also may be used for thefirst pretreatment step. By alkaline is meant a pH of greater than 7.0.Particularly suitable is a pH of the biomass-aqueous ammonia mixturethat is greater than 8. In one embodiment, ammonia is present at lessthan about 10 weight percent relative to dry weight of biomass.Particularly suitable is ammonia at less than about 6 weight percentrelative to dry weight of biomass. In some embodiments, ammonia isselected from the group consisting of ammonia gas, ammonium hydroxide,urea, and combinations thereof.

Ammonia as used in one step of the present methods provides advantagesover other bases. Ammonia partitions into a liquid phase and a vaporphase. Gaseous ammonia can diffuse more easily through biomass than aliquid base, resulting in more efficacious pretreatment at lowerconcentrations. Ammonia also is known to compete with hydrolysis, viaammonolysis, of acetyl esters in biomass to form acetamide.

Acetamide is less toxic than acetate to certain fermentation organisms,such as Zymomonas mobilis. See, for example, published patentapplication US 2007/0031918. Thus conversion of acetyl esters toacetamide rather than to acetic acid reduces the need to remove aceticacid. The use of ammonia also reduces the requirement to supplementgrowth medium used during fermentation with a nitrogen source.

In addition, ammonia is a low-cost material and thus provides aneconomical process. Ammonia can also be recycled to the pretreatmentreactor during pretreatment or following pretreatment, thus enabling amore economical process. For example, following pretreatment withammonia, as the temperature is decreased to that suitable for ozonationor saccharification, ammonia gas may be released, optionally in thepresence of a vacuum, and may be recycled. In a continuous process,ammonia may be continuously recycled.

The aqueous solution comprising ammonia may optionally comprise at leastone additional base, such as sodium hydroxide, sodium carbonate,potassium hydroxide, potassium carbonate, calcium hydroxide and calciumcarbonate. The at least one additional base may be added in an amountthat is combined with ammonium to form an amount of total base that isless than about 20 weight percent relative to biomass dry weight.Preferably the total second base plus ammonia is in an amount that isless than about 15 weight percent. Additional base(s) may be utilized,for example, to neutralize acids in biomass, to provide metal ions forthe saccharification enzymes, or to provide metal ions for thefermentation growth medium.

In the present methods, the biomass dry weight is at an initialconcentration of at least about 15% up to about 80% of the weight of thebiomass-aqueous ammonia mixture. More suitably, the dry weight ofbiomass is at a concentration of from about 15% to about 60% of theweight of the biomass-aqueous ammonia mixture. The percent of biomass inthe biomass-aqueous ammonia mixture is kept high to minimize the totalvolume of pretreatment material, making the process more economical.Keeping the percent biomass high also reduces the need for concentrationof sugars resulting from saccharification of the pretreated biomass, foruse in fermentation.

Pretreatment of biomass with ammonia solution may be carried out in anysuitable vessel. Typically the vessel is one that can withstandpressure, has a mechanism for heating, and has a mechanism for mixingthe contents. Commercially available vessels include, for example, theZipperclave® reactor (Autoclave Engineers, Erie, Pa.), the Jaygo reactor(Jaygo Manufacturing, Inc., Mahwah, N.J.), and a steam gun reactor((described in General Methods Autoclave Engineers, Erie, Pa.). Muchlarger scale reactors with similar capabilities may be used.Alternatively, the biomass and ammonia solution may be combined in onevessel, then transferred to another reactor. Also biomass may bepretreated in one vessel, then further processed in another reactor suchas a steam gun reactor (described in General Methods; AutoclaveEngineers, Erie, Pa.).

Prior to contacting the biomass with an aqueous solution comprisingammonia, vacuum may be applied to the vessel containing the biomass. Byevacuating air from the pores of the biomass, better penetration of thesolvent into the biomass may be achieved. The time period for applyingvacuum and the amount of negative pressure that is applied to thebiomass will depend on the type of biomass and can be determinedempirically so as to achieve optimal pretreatment of the biomass (asmeasured by the production of fermentable sugars followingsaccharification).

The contacting of the biomass with an aqueous solution comprisingammonia may be carried out at a temperature of from about 4° C. to about200° C. Initial contacting of the biomass with ammonia at 4° C.,allowing impregnation at this temperature, was found to increase theefficiency of saccharification over non-pretreated native biomass. Inanother embodiment, contacting of the biomass may be carried out at atemperature of from about 75° C. to about 150° C. In still anotherembodiment, contacting of the biomass may be carried out at atemperature of from greater than about 90° C. to about 150° C.

The contacting of the biomass with an aqueous solution comprisingammonia may be carried out for a period of time up to about 25 hours.Longer periods of pretreatment are possible, however a shorter period oftime may be preferable for practical, economic reasons. Typically aperiod of ammonia contact treatment may be about 8 hours or less. Longerperiods may provide the benefit of reducing the need for application ofenergy for breaking up the biomass, therefore, a period of time up toabout 25 hours may be preferable.

In one embodiment, the ammonia treatment step may be performed at arelatively high temperature for a relatively short period of time, forexample at from about 100° C. to about 150° C. for about 5 minutes toabout 2 hours. In another embodiment, the ammonia treatment step may beperformed at a lower temperature for a relatively long period of time,for example from about 75° C. to about 100° C. for about 2 hours toabout 8 hours. In still another embodiment, the pretreatment process maybe performed at room temperature for an even longer period of time ofabout 24 hours. Other temperature and time combinations intermediate tothese may also be used.

For the treatment with aqueous ammonia solution, the temperature, timefor pretreatment, ammonia concentration, concentration of one or moreadditional reagents, biomass concentration, biomass type and biomassparticle size are related; thus these variables may be adjusted asnecessary to obtain an optimal product to be contacted with a gascomprising ozone or with a saccharification enzyme consortium, dependingon the pretreatment method used.

The treatment with aqueous ammonia solution may be performed in anysuitable vessel, such as a batch reactor or a continuous reactor. Oneskilled in the art will recognize that at higher temperatures (above100° C.), a pressure vessel is required. The suitable vessel may beequipped with a means, such as impellers, for agitating thebiomass-aqueous ammonia mixture. Reactor design is discussed in Lin,K.-H., and Van Ness, H. C. (in Perry, R. H. and Chilton, C. H. (eds),Chemical Engineer's Handbook, 5^(th) Edition (1973) Chapter 4,McGraw-Hill, NY). The pretreatment reaction may be carried out as abatch process, or as a continuous process.

It is well known to those skilled in the art that a nitrogen source isrequired for growth of microorganisms during fermentation; thus the useof ammonia during pretreatment provides a nitrogen source and reduces oreliminates the need to supplement the growth medium used duringfermentation with a nitrogen source. If the pH of the pretreatmentproduct exceeds that at which saccharification enzymes are active, orexceeds the range suitable for microbial growth in fermentation, acidsmay be utilized to reduce pH. The amount of acid used to achieve thedesired pH may result in the formation of salts at concentrations thatare inhibitory to saccharification enzymes or to microbial growth. Inorder to reduce the amount of acid required to achieve the desired pHand to reduce the raw material cost of NH₃ in the present pretreatmentprocess, ammonia gas may be evacuated from the pretreatment reactor andrecycled. Typically, at least a portion of the ammonia is removed, whichreduces the pH but leaves some nitrogen that provides this nutrient foruse in subsequent fermentation.

Alternatively, performing ozonation after ammonia pretreatment has itsadvantages, too. Ammonia is an inhibitor of hydrolytic enzymes-ozonationof ammonia-pretreated biomass will result in removal of residualammonia, as ozone reacts with ammonia to yield nitrogen or nitrate. Theresulting biomass would thus require less acid for adjusting the pH forsubsequent steps (saccharification and fermentation). Typically, afterammonia treatment the biomass contains about 40 percent to about 60percent moisture. If the biomass is dry, water may be added to adjustthe moisture content to between about 30 percent and about 60 percent.

In order to obtain sufficient quantities of sugars from biomass, thebiomass may be pretreated with an aqueous ammonia solution one time ormore than one time. Similarly, an ozonation step or a saccharificationreaction may be performed one or more times. Both pretreatment andsaccharification processes may be repeated if desired to obtain higheryields of sugars. To assess performance of the pretreatment andsaccharification processes, separately or together, the theoreticalyield of sugars derivable from the starting biomass can be determinedand compared to measured yields.

Ozone Treatment:

According to the present methods, lignocellulosic biomass orammonia-pretreated biomass is contacted with a gas comprising ozone.Ozone treatment promotes oxidation and fragmentation of the lignin andis beneficial to pretreatment, resulting in an increased accessibilityof the carbohydrate-enriched biomass to enzymatic saccharification. Theuse of ozone as a means of lignin removal is relatively selective,leaving the carbohydrates largely intact. In addition, ozone (O₃) easilydecomposes to oxygen (O₂) and water, leaving no residue from its use andcontributing minimal atmospheric pollution.

The ozone may be generated by any means known in the art, for examplefrom oxygen or air. In the present methods, the gas comprising ozonecomprises about 0.1 to about 20 percent by volume ozone, for exampleabout 0.5 to about 10 percent by volume ozone. The gas may furthercomprise nitrogen, oxygen, argon, or a combination thereof. The gascomprising ozone may also comprise one or more other gases as long asthe presence or concentration of the other gases is not deleterious tothe ozone treatment. Generally, the ratio of ozone to the pretreatedbiomass may be at least about 1:1200 on a weight basis, for example forexample at least about 1:1000, or at least about 1:750, or at leastabout 1:500, or at least about 1:200, or at least about 1:100, or atleast about 1:50.

In one method, lignocellulosic biomass is contacted with an aqueoussolution comprising ammonia, then the ammonia-treated biomass iscontacted with a gas comprising ozone. In some embodiments, the ratio ofozone to the ammonia-treated biomass may be at least about 1:500 on aweight basis, for example at least about 2 mg of ozone per gram ofbiomass. In some embodiments the ratio of ozone to the ammonia-treatedbiomass may be at least about 1:100. In some embodiments, the amount ofozone in relation to the pretreated biomass may be from about 0.2 mgO₃/g biomass to about 10 mg O₃/g biomass. Other ratios may also be used.Preferred is a ratio of ozone to biomass which is sufficient to fragmentlignin while retaining carbohydrate in good yield. Use of excess ozonebeyond that which is optimal for delignification may lead tocarbohydrate loss and lower sugar yields through saccharification.

In one method, lignocellulosic biomass is contacted with a gascomprising ozone, then the ozone-treated biomass is contacted with anaqueous solution comprising ammonia. In some embodiments, the ratio ofozone to the native lignocellulosic biomass may be at least about 1:100on a weight basis, for example at least about 10 mg of ozone per gram ofbiomass. In some embodiments, the ratio of ozone to the lignocellulosicbiomass may be from about 1:100 to about 1:50. Lower ratios may also beused. Preferred is a ratio of ozone to biomass which is sufficient tofragment lignin while retaining carbohydrate in good yield. Use ofexcess ozone beyond that which is optimal for delignification may leadto carbohydrate loss and lower sugar yields through saccharification.

Ozone Treatment Conditions:

Contacting of the native lignocellulosic biomass or theammonia-pretreated biomass with a gas comprising ozone may be carriedout in any suitable vessel, such as a batch reactor or a continuousreactor. Typically the vessel is one that has a mechanism for heating orcooling, and has a mechanism for mixing the contents. Optionally, thevessel is one that can withstand pressure. The ozone pretreatmentreaction may be performed in a fixed bed reactor, for example, or in arotating horizontal cylinder, or a continuous stirred tank reactor. Thesuitable vessel may be equipped with a means, such as impellers, foragitating the biomass or the aqueous biomass suspension, or the vesselitself may rotate or spin to agitate the solid biomass. Reactor designis discussed in Lin, K.-H., and Van Ness, H. C. (in Perry, R. H. andChilton, C. H. (eds), Chemical Engineer's Handbook, 5^(th) Edition(1973) Chapter 4, McGraw-Hill, NY). The ozone pretreatment reaction maybe carried out as a batch process, or as a continuous process. Thebiomass may be contacted with ozone in the same reactor as theammonia-treatment is performed, or in another reactor. The biomass maybe contacted with ozone in one reactor, then saccharified in the samevessel; alternatively, saccharification may be performed in a separatevessel.

Prior to contacting the biomass with a gas comprising ozone, the nativelignocellulosic biomass or the ammonia-pretreated biomass may be driedby conventional means. The dried native lignocellulosic orammonia-pretreated biomass may contain about 10 percent to about 70percent moisture, for example from about 30 percent to about 60 percentmoisture.

Contacting the biomass with a gas comprising ozone may be carried out ata temperature of from about 0° C. to about 50° C. In one embodiment, thetemperature may be from about 0° C. to about 25° C. Higher temperaturesmay be used but are generally less practical as ozone decompositionincreases with increasing temperature. Lower temperatures may also beused but are generally less economical due to cooling requirements and,in the case where an aqueous biomass suspension is used, may not bepractical from an operability standpoint. Contacting the biomass with agas comprising ozone may be carried out for a reaction time of at leastabout 1 minute, for example for a reaction time of about 1 minute toabout 60 minutes, or about 1 minute to about 30 minutes, or about 1minute to about 25 minutes, or about 1 minute to about 20 minutes, orabout 1 minute to about 15 minutes, or about 1 minute to about 10minutes, or about 1 minute to about 5 minutes. Extending the ozonationtime beyond that optimal for lignin degradation may result in decreasedsugar yields, presumably due to sugar degradation.

Contacting the biomass with a gas comprising ozone may be performed atautogeneous pressure. Higher or lower pressures may also be used but aregenerally less practical.

The biomass may be contacted in the solid state with the gas comprisingozone, without a liquid phase being present. Alternatively, the biomassmay be contacted as an aqueous suspension with the gas comprising ozone.To generate a biomass suspension, ammonia-treated biomass is contactedwith an aqueous solution. The weight percent of biomass in the aqueousammonia-treated biomass suspension can be from about 20 weight percentto about 70 weight percent, for example from about 30 weight percent toabout 60 weight percent. The aqueous ammonia-treated biomass suspensioncan have a pH of about 1 to about 10, for example from about 2 to about9, or from about 1 to about 7, or from about 1 to about 5. The aqueoussolution may further comprise a buffer, for example a citrate buffer.The selection of an appropriate buffer may be based on the buffer'ssuitability for controlling pH in a subsequent saccharification. Afterozone treatment is complete, the pH of the aqueous biomass suspensioncan be adjusted to a second pH sufficient for enzymatic saccharificationof the biomass, if desired.

For the ozone pretreatment step, the temperature, time for pretreatment,ozone concentration in the gas, moisture content, biomass concentration,ratio of ozone to biomass, biomass type, and biomass particle size arerelated; thus these variables may be adjusted as necessary for each typeof biomass to optimize the pretreatment processes described herein.

To assess performance of the pretreatment, i.e., the production ofreadily saccharifiable biomass, and subsequent saccharification,separately or together, the theoretical yield of sugars derivable fromthe starting biomass may be determined and compared to measured yields.

Further Processing:

Saccharification:

Following pretreatments of ozone followed by aqueous ammonia, or aqueousammonia followed by ozone, the readily saccharifiable biomass comprisesa mixture of fragmented lignin and polysaccharides. If desired, prior tofurther processing, the lignin fragments or oxidation products may beremoved from the pretreated biomass by filtering and optionally washingthe sample with EtOH/H₂O (0% to 100% EtOH volume/volume [v/v]). As thefiltration and washing steps are not necessary to obtain improved sugaryields, and as the costs associated with them may negatively impact theeconomics of the method, filtering and washing of the biomass ispreferably omitted. The biomass may be dried at room temperature,resulting in readily saccharifiable biomass. The concentration ofglucan, xylan and acid-insoluble lignin content of the readilysaccharifiable biomass may be determined using analytical means wellknown in the art.

The readily saccharifiable biomass may then be further hydrolyzed in thepresence of a saccharification enzyme consortium to releaseoligosaccharides and/or monosaccharides in a hydrolysate. Surfactantssuch as polyethylene glycols (PEG) may be added to improve thesaccharification process (U.S. Pat. No. 7,354,743 B2, incorporatedherein by reference). Saccharification enzymes and methods for biomasstreatment are reviewed in Lynd, L. R., et al. (Microbiol. Mol. Biol.Rev., 66:506-577, 2002). The saccharification enzyme consortium maycomprise one or more glycosidases; the glycosidases may be selected fromthe group consisting of cellulose-hydrolyzing glycosidases,hemicellulose-hydrolyzing glycosidases, and starch-hydrolyzingglycosidases. Other enzymes in the saccharification enzyme consortiummay include peptidases, lipases, ligninases and feruloyl esterases.

The saccharification enzyme consortium comprises one or more enzymesselected primarily, but not exclusively, from the group “glycosidases”which hydrolyze the ether linkages of di-, oligo-, and polysaccharidesand are found in the enzyme classification EC 3.2.1.x (EnzymeNomenclature 1992, Academic Press, San Diego, Calif. with Supplement 1(1993), Supplement 2 (1994), Supplement 3 (1995, Supplement 4 (1997) andSupplement 5 [in Eur. J. Biochem., 223:1-5, 1994; Eur. J. Biochem.,232:1-6, 1995; Eur. J. Biochem., 237:1-5, 1996; Eur. J. Biochem.,250:1-6, 1997; and Eur. J. Biochem., 264:610-650 1999, respectively]) ofthe general group “hydrolases” (EC 3). Glycosidases useful in thepresent method can be categorized by the biomass component that theyhydrolyze. Glycosidases useful for the present method includecellulose-hydrolyzing glycosidases (for example, cellulases,endoglucanases, exoglucanases, cellobiohydrolases, β-glucosidases),hemicellulose-hydrolyzing glycosidases (for example, xylanases,endoxylanases, exoxylanases, β-xylosidases, arabino-xylanases, mannases,galactases, pectinases, glucuronidases), and starch-hydrolyzingglycosidases (for example, amylases, α-amylases, β-amylases,glucoamylases, α-glucosidases, isoamylases). In addition, it may beuseful to add other activities to the saccharification enzyme consortiumsuch as peptidases (EC 3.4.x.y), lipases (EC 3.1.1.x and 3.1.4.x),ligninases (EC 1.11.1.x), and feruloyl esterases (EC 3.1.1.73) to helprelease polysaccharides from other components of the biomass. It is wellknown in the art that microorganisms that producepolysaccharide-hydrolyzing enzymes often exhibit an activity, such ascellulose degradation, that is catalyzed by several enzymes or a groupof enzymes having different substrate specificities. Thus, a “cellulase”from a microorganism may comprise a group of enzymes, all of which maycontribute to the cellulose-degrading activity. Commercial ornon-commercial enzyme preparations, such as cellulase, may comprisenumerous enzymes depending on the purification scheme utilized to obtainthe enzyme. Thus, the saccharification enzyme consortium of the presentmethod may comprise enzyme activity, such as “cellulase”, however it isrecognized that this activity may be catalyzed by more than one enzyme.

Saccharification enzymes may be obtained commercially, in isolated form,such as Spezyme® CP cellulase (Genencor International, Rochester, N.Y.)and Multifect® xylanase (Genencor). In addition, saccharificationenzymes may be expressed in host organisms at the biofuels plant,including using recombinant microorganisms.

One skilled in the art would know how to determine the effective amountof enzymes to use in the consortium and adjust conditions for optimalenzyme activity. One skilled in the art would also know how to optimizethe classes of enzyme activities required within the consortium toobtain optimal saccharification of a given pretreatment product underthe selected conditions.

Preferably the saccharification reaction is performed at or near thetemperature and pH optima for the saccharification enzymes. Thetemperature optimum used with the saccharification enzyme consortium inthe present method ranges from about 15° C. to about 100° C. In anotherembodiment, the temperature optimum ranges from about 20° C. to about80° C. Most typically the temperature optimum ranges from about 45° C.to about 50° C. The pH optimum can range from about 2 to about 11. Inanother embodiment, the pH optimum used with the saccharification enzymeconsortium in the present method may range from about 4 to about 5.5.

The saccharification may be performed for a time of about severalminutes to about 120 hours, and preferably from about several minutes toabout 48 hours. The time for the reaction will depend on enzymeconcentration and specific activity, as well as the substrate used andthe environmental conditions, such as temperature and pH. One skilled inthe art can readily determine optimal conditions of temperature, pH andtime to be used with a particular substrate and saccharificationenzyme(s) consortium.

The saccharification may be performed batch-wise or as a continuousprocess. The saccharification may also be performed in one step, or in anumber of steps. For example, different enzymes required forsaccharification may exhibit different pH or temperature optima. Aprimary treatment may be performed with enzyme(s) at one temperature andpH, followed by secondary or tertiary (or more) treatments withdifferent enzyme(s) at different temperatures and/or pH. In addition,treatment with different enzymes in sequential steps may be at the samepH and/or temperature, or different pHs and temperatures, such as usinghemicellulases stable and more active at higher pHs and temperaturesfollowed by cellulases that are active at lower pHs and temperatures.

The degree of solubilization of sugars from biomass followingsaccharification may be monitored by measuring the release ofmonosaccharides and oligosaccharides. Methods to measure monosaccharidesand oligosaccharides are well known in the art. For example, theconcentration of reducing sugars can be determined using the1,3-dinitrosalicylic (DNS) acid assay (Miller, G. L., Anal. Chem., 31:426-428, 1959). Alternatively, sugars can be measured by HPLC using anappropriate column as described below.

Fermentation to Target Products:

The readily saccharifiable biomass produced by the present methods maybe hydrolyzed by enzymes as described above to produce fermentablesugars which then can be fermented into a target product. “Fermentation”refers to any fermentation process or any process comprising afermentation step. Target products include, without limitation alcohols(e.g., arabinitol, butanol, ethanol, glycerol, methanol,1,3-propanediol, sorbitol, and xylitol); organic acids (e.g., aceticacid, acetonic acid, adipic acid, ascorbic acid, citric acid,2,5-diketo-D-gluconic acid, formic acid, fumaric acid, glucaric acid,gluconic acid, glucuronic acid, glutaric acid, 3-hydroxypropionic acid,itaconic acid, lactic acid, malic acid, malonic acid, oxalic acid,propionic acid, succinic acid, and xylonic acid); ketones (e.g.,acetone); amino acids (e.g., aspartic acid, glutamic acid, glycine,lysine, serine, and threonine); gases (e.g., methane, hydrogen (H₂),carbon dioxide (CO₂), and carbon monoxide (CO)).

Fermentation processes also include processes used in the consumablealcohol industry (e.g., beer and wine), dairy industry (e.g., fermenteddairy products), leather industry, and tobacco industry.

Further to the above, the sugars produced from saccharifying thepretreated biomass as described herein may be used to produce ingeneral, organic products, chemicals, fuels, commodity and specialtychemicals such as xylose, acetone, acetate, glycine, lysine, organicacids (e.g., lactic acid), 1,3-propanediol, butanediol, glycerol,ethylene glycol, furfural, polyhydroxyalkanoates, cis, cis-muconic acid,and animal feed (Lynd, L. R., Wyman, C. E., and Gerngross, T. U.,Biocommodity Engineering, Biotechnol. Prog., 15: 777-793, 1999; andPhilippidis, G. P., Cellulose bioconversion technology, in Handbook onBioethanol: Production and Utilization, Wyman, C. E., ed., Taylor &Francis, Washington, D.C., 179-212, 1996; and Ryu, D. D. Y., andMandels, M., Cellulases: biosynthesis and applications, Enz. Microb.Technol., 2: 91-102, 1980).

Potential coproducts may also be produced, such as multiple organicproducts from fermentable carbohydrate. Lignin-rich residues remainingafter pretreatment and fermentation can be converted to lignin-derivedchemicals, chemical building blocks or used for power production.

Conventional methods of fermentation and/or saccharification are knownin the art including, but not limited to, saccharification,fermentation, separate hydrolysis and fermentation (SHF), simultaneoussaccharification and fermentation (SSF), simultaneous saccharificationand cofermentation (SSCF), hybrid hydrolysis and fermentation (HHF), anddirect microbial conversion (DMC).

SHF uses separate process steps to first enzymatically hydrolyzecellulose to sugars such as glucose and xylose and then ferment thesugars to ethanol. In SSF, the enzymatic hydrolysis of cellulose and thefermentation of glucose to ethanol is combined in one step (Philippidis,G. P., in Handbook on Bioethanol: Production and Utilization, Wyman, C.E., ed., Taylor & Francis, Washington, D.C., 179-212, 1996). SSCFincludes the cofermentation of multiple sugars (Sheehan, J., and Himmel,M., Bioethanol, Biotechnol. Prog. 15: 817-827, 1999). HHF includes twoseparate steps carried out in the same reactor but at differenttemperatures, i.e., high temperature enzymatic saccharification followedby SSF at a lower temperature that the fermentation strain can tolerate.DMC combines all three processes (cellulase production, cellulosehydrolysis, and fermentation) in one step (Lynd, L. R., Weimer, P. J.,van Zyl, W. H., and Pretorius, I. S., Microbiol. Mol. Biol. Reviews, 66:506-577, 2002).

These processes may be used to produce target products from the readilysaccharifiable biomass produced by the pretreatment methods describedherein.

Advantages of the Present Methods:

One of the advantages of the present methods is the high selectivity forfragmenting and removing lignin from the biomass while leaving thecarbohydrates largely intact. Less selective pretreatment methodshydrolyze a portion of the carbohydrates to sugars, for example aportion of the glucans to glucose and/or a portion of the xylans toxylose. If present, the monomeric sugars can be degraded during thepretreatment process, resulting in a decrease in the overall yield tosugar (i.e. through a saccharification step). As demonstrated by theExamples, prolonged ozonation can lead to diminished yields of sugars,in particular xylose. Therefore, there exists an optimal reaction timefor ozone treatment, below which the pretreatment will be ineffective,and above which it will be unselective. The optimal reaction time forozone treatment depends in part on the biomass composition, inparticular lignin content, the particle size, and the amount of ozoneused relative to the biomass.

Another advantage of the present methods is that separation or washingof the biomass after ozone treatment to physically remove the oxidizedand fragmented lignin is not necessary. The monomeric sugars, being moresoluble than cellulose and hemicellulose, can be separated from thecarbohydrates when filtration and washing of the treated biomass arenecessary before saccharification, resulting in a decrease in theoverall yield to sugar. The present methods minimize sugar loss duringlignin oxidation and fragmentation, which is of economic benefit.

In particular, the present methods provide surprisingly good xyloserecovery through saccharification. Xylose recovery can be substantiallylower than glucose recovery, when compared to the theoretical yields ofthe sugars based on the total amount of sugars present in the nativebiomass before any pretreatment. This arises from the vast difference inthe kinetics of hydrolysis of xylans and glucans, which are moredifficult and easier to hydrolyze, respectively. It was not expectedthat xylose recovery would be as high as seen with the present methodsusing optimal reaction conditions. Upon ozone treatment, the lignin,hemicellulose, and cellulose content of the biomass is decreased, withlignin being the most severely affected, followed by hemicellulose andcellulose, respectively. The present methods provide conditions underwhich lignin is selectively degraded in the presence of hemicelluloseand cellulose, without negatively affecting their saccharificationyields. This is especially significant in the case of xylose yield, ashemicellulose is more easily degraded with ozone.

Additionally, lignin is more electron rich than the carbohydratescontained in biomass, and as a result the lignin is more prone tooxidation by the ozone than are the carbohydrates. While not wishing tobe bound by any theory, oxidation of the lignin by the ozone is believedto reduce the molecular weight of the lignin fragments, which in turnrenders the lignin fragments both more soluble in the solvent solutionand less able to bind to cellulolytic enzymes. As a result, the use oflower enzyme loadings in saccharification is enabled, which can providecost savings with regard to enzyme usage. The present methodsadvantageously combine the use of pretreatment with an aqueous solutioncomprising ammonia followed by selective oxidation of lignin by ozonetreatment to produce a readily saccharifiable biomass.

The present methods offer advantageous flexibility regarding ozonationin that the ozone treatment may be performed on solid biomass or on anaqueous suspension of biomass. Both options offer opportunity foroverall process simplification and economic benefit. For example, ifdesired the biomass may be treated with ozone as an aqueous biomasssuspension, wherein the suspension is formed from an aqueous solutioncomprising a buffer selected for a subsequent saccharification step.After ozone treatment, the enzyme cocktail may be added directly to thereadily saccharifiable biomass and saccharification can be performed inthe same reaction vessel. Alternatively, solid biomass may be contactedwith ozone, that is, without the presence of a liquid phase.

EXAMPLES

The goal of the experimental work described below was to develop apretreatment process for lignocellulose that maximized lignindegradation and minimized carbohydrate loss in the pretreatment toproduce a readily saccharifiable biomass that may be further processedto result in a maximal monomeric sugar yield following enzymaticsaccharification. The approach adopted was to selectively oxidize andfragment the lignin in the presence of a gas comprising ozone whileretaining the sugars with the solids residue. The following experimentsshow that ozone treatment of ammonia-pretreated biomass oxidized andfragmented the lignin to produce a readily saccharifiable biomass.

The present invention is further defined in the following examples. Itshould be understood that these examples, while indicating preferredembodiments of the invention, are given by way of illustration only.From the above discussion and these examples, one skilled in the art canascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various uses andconditions.

The following materials were used in the examples. All commercialreagents were used as received.

Glucose, xylose, cellobiose, and citric acid were obtained fromSigma-Aldrich (St. Louis, Mo.). Suitable zirconium pellets can beobtained from Union Process (Akron, Ohio) or Ortech Advanced Ceramics(Sacramento, Calif.).

Corn cob was obtained from University of Wisconsin Farm, in Madison,Wis. and was milled to assorted sizes. Switchgrass was obtained fromGenera Energy. The switchgrass sample particles were less than 1 mm insize, and the initial moisture content was about 7 weight percent.

Carbohydrate Analysis of Biomass

A modified version of the NREL LAP procedure “Determination ofStructural Carbohydrates and Lignin in Biomass” was used to determinethe weight percent glucan and xylan in the biomass. Sample preparationwas simplified by drying at 80° C. under vacuum or at 105° C. underambient pressure overnight. The samples were knife milled to passthrough a 20 mesh screen but were not sieved. The dry milled solids werethan subjected to the acid hydrolysis procedure at a 50 mg solids scale.The solids were not first extracted with water or ethanol. HPLC analysisof sugars was done on an Aminex HPX-87H column and no analysis of ligninwas attempted.

The soluble sugars glucose, cellobiose, and xylose in saccharificationliquor were measured by HPLC (Agilent 1100, Santa Clara, Calif.) usingBio-Rad HPX-87H column (Bio-Rad Laboratories, Hercules, Calif.) withappropriate guard columns, using 0.01 N aqueous sulfuric acid as theeluant. The sample pH was measured and adjusted to 5-6 with sulfuricacid if necessary. The sample was then passed through a 0.2 μm syringefilter directly into an HPLC vial. The HPLC run conditions were asfollows:

-   -   Biorad Aminex HPX-87H (for carbohydrates):    -   Injection volume: 10-50 μL, dependent on concentration and        detector limits    -   Mobile phase: 0.01 N aqueous sulfuric acid, 0.2 micron filtered        and degassed    -   Flow rate: 0.6 mL/minute    -   Column temperature: 50° C., guard column temperature <60° C.    -   Detector temperature: as close to main column temperature as        possible    -   Detector: refractive index    -   Run time: 15 minute data collection        After the run, concentrations in the sample were determined from        standard curves for each of the compounds.

Ozone was generated from air using an ozonizer (model CD1500)manufactured by ClearWater Tech (San Luis Obispo, Calif.) and set onmaximum voltage. The amount of ozone used in each Example with anozonation step was calculated from the ozone consumed during theindicated reaction time by measuring the ozone concentration in theozone-enriched air entering and leaving the experimental apparatus andtaking the difference. Ozone measurements were made using a TeledyneInstruments (San Diego, Calif.) ozone monitor, model 450 M.

The moisture content of the biomass was determined by the NationalRenewable Energy Laboratory (NREL) procedure “Determination of TotalSolids in Biomass and Total Dissolved Solids in Liquid Process Samples”.

The roll mill was manufactured by US Stoneware (East Palestine, Ohio).

The following abbreviations are used:

“HPLC” is High Performance Liquid Chromatography, “C” is degreesCentigrade or Celsius; “%” is percent; “mL” is milliliter; “h” ishour(s); “rpm” is revolution per minute; “EtOH” is ethanol; “mg/g” ismilligram per gram; “g/100 mL” is gram per 100 milliliter; “g” is gram;“NaOH” is sodium hydroxide; “w/v” is weight per volume; “v/v” is volumefor volume, “w/w” is weight for weight; “mm” is millimeter; “mL/min” ismilliliter per minute; “min” is minutes; “mM” is millimolar, “N” isnormal, “μL” is microliter.

The biomass used in Examples 1-6 and Comparative Examples A and B waspretreated with an aqueous solution comprising ammonia according to thefollowing procedure. The same procedure was used in Examples 7-15 andComparative Examples C through H, except that switchgrass was used inplace of corn cob and ammonia treatment was the second or onlypretreatment step.

To a 170 L jacketed horizontal paddle reactor (Jaygo Manufacturing, Inc,Mahwah, N.J.) were added corn cobs (25.7 kg). Vacuum was then applied tothe reactor to reach a pressure of −10.5 psig. After that, an aqueoussolution of ammonia (14.88 kg of 7.63 wt % of ammonia) was added to thereactor, followed by water (1.58 kg). Steam was then injected to bringthe reactor temperature to 145° C. for 20 minutes. The reactor was thenvented to atmospheric pressure, and the mixture was then evacuated undervacuum to a pressure of −10.5 psig. Sterile air was then introduced tothe reactor, the pretreated cob was collected and ground using a hammermill, and sieved through a ½ inch screen.

Examples 1 through 3 and Examples 4 through 6 illustrate a method forproducing readily saccharifiable biomass by pretreatment with diluteammonia followed by ozonation of the biomass as an aqueous suspension.The effect of the pretreatment was quantified by saccharifying thereadily saccharifiable biomass to determine the theoretical yields forglucose and xylose. Theoretical yields encompass the monomeric sugaryields obtained through the pretreatment and saccharification stepscompared to the total amount of sugars present in the native biomassbefore any pretreatment was performed. Biomass samples from Examples 1-3were saccharified using Spezyme, Novo 188, and Multifect enzymecocktails; Examples 4-6 were saccharified at lower enzyme loading, andwith Accellerase/Multifect enzyme cocktails.

To demonstrate the beneficial effect of the ozonation, ComparativeExample A was performed as a control experiment following the sameprocedure as Examples 1-3 but without the ozone treatment step. Theammonia-pretreated biomass of Comparative Example A was saccharifiedusing Spezyme, Novo 188, and Multifect enzyme cocktails. As anothercontrol experiment, Comparative Example B was performed following thesame procedure as Examples 4-6 but without the ozone treatment step. Theammonia-pretreated biomass of Comparative Example B was saccharified atlower enzyme loading and with Accellerase/Multifect enzyme cocktails.

For Examples 1-3 and Comparative Example A, theoretical yields forglucose and xylose are given in Tables 1 and 2, respectively. ForExamples 4-6 and Comparative Example B, theoretical yields for glucoseand xylose are given in Tables 3 and 4, respectively.

Example 1 Effect of Ozonation of an Aqueous Ammonia-Treated BiomassSuspension for 10 Minutes

To a slurry of ammonia-pretreated corn cob (6.0 g of 60% dry solid, 3.6g dry solid) in citrate buffer (19.36 mL, pH=5) was introduced a streamof ozone-enriched air (flow rate 2 L/min) at room temperature. After 10minutes and the consumption of 5.0 mg of ozone, the flow ofozone-enriched air was stopped and the slurry was charged with Spezyme(150.0 μL, concentration 168.5 mg/mL), Multifect (180.0 μL,concentration 56.1 mg/mL), and Novozyme 188 (25.0 μL, concentration 253mg/mL) enzyme cocktails, and the mixture was left stirring in anincubator/shaker at 48° C. Samples were taken every 24 h and analyzed byHPLC to determine the monomeric sugar yields versus time. Results areshown in Tables 1 and 2.

Example 2 Effect of Ozonation of an Aqueous Ammonia-Treated BiomassSuspension for 20 Minutes

To a slurry of ammonia-pretreated corn cob (6.0 g of 60% dry solid, 3.6g dry solid) in citrate buffer (19.36 mL, pH=5) was introduced a streamof ozone-enriched air (flow rate 2 L/min) at room temperature. After 20minutes and the consumption of 10.0 mg of ozone, the flow ofozone-enriched air was stopped and the slurry was charged with Spezyme(150.0 μL, concentration 168.5 mg/mL), Multifect (180.0 μL,concentration 56.1 mg/mL), and Novozyme 188 (25.0 μL, concentration 253mg/mL) enzyme cocktails, and the mixture was left stirring in anincubator/shaker at 48° C. Samples were taken every 24 h and analyzed byHPLC to determine the monomeric sugar yields versus time. Results areshown in Tables 1 and 2.

Example 3 Effect of Ozonation of an Aqueous Ammonia-Treated BiomassSuspension for 30 Minutes

To a slurry of ammonia-pretreated corn cob (6.0 g of 60% dry solid, 3.6g dry solid) in citrate buffer (19.36 mL, pH=5) was introduced a streamof ozone-enriched air (flow rate 2 L/min) at room temperature. After 30minutes and the consumption of 15.1 mg of ozone, the flow ofozone-enriched air was stopped and the slurry was charged with Spezyme(150.0 μL, concentration 168.5 mg/mL), Multifect (180.0 μL,concentration 56.1 mg/mL), and Novozyme 188 (25.0 μL, concentration 253mg/mL) enzyme cocktails, and the mixture was left stirring in anincubator/shaker at 48° C. Samples were taken every 24 h and analyzed byHPLC to determine the monomeric sugar yields versus time. Results areshown in Tables 1 and 2.

Comparative Example A Control Experiment with No Ozonation

To a slurry of ammonia-pretreated corn cob (6.0 g of 60% dry solid, 3.6g dry solid) in citrate buffer (19.36 mL, pH=5) was added Spezyme (150.0μL, concentration 168.5 mg/mL), Multifect (180.0 μL, concentration 56.1mg/mL), and Novozyme 188 (25.0 μL, concentration 253.0 mg/mL) enzymecocktails, and the mixture was left stirring in an incubator/shaker at48° C. Samples were taken every 24 h and analyzed by HPLC to determinethe monomeric sugar yields versus time. Results are shown in Tables 1and 2.

TABLE 1 Theoretical yields for glucose during saccharification ofbiomass from Examples 1-3 and Comparative Example A. SaccharificationComparative Time (hours) Example A Example 1 Example 2 Example 3 0 0 0 00 24 32.1 39.9 38.8 42.2 48 35.1 51.2 53.4 47.9 72 39.3 58.1 55.6 49.396 41.3 66.1 56.2 49.6 120 45.1 68.7 63.5 51.5

TABLE 2 Theoretical yields for xylose during saccharification of biomassfrom Examples 1-3 and Comparative Example A. SaccharificationComparative Time (hours) Example A Example 1 Example 2 Example 3 0 0 0 00 24 24.7 32.9 19.1 29.7 48 29.4 40.5 25.2 31.8 72 34.0 42.0 26.1 33.096 44.8 50.4 26.4 33.8 120 45.4 50.8 30.3 36.9

As shown in Table 1, ozone treatment of the ammonia-treated corn cobprovided readily saccharifiable biomass which provided improved glucoseyields upon saccharification. Higher glucose yields were observed withlonger ozone treatment reaction times. The data in Table 2 indicatesthat there was an optimal reaction time for ozone treatment and xyloserecovery. With longer reaction times for ozone treatment, xylose wasdegraded and xylose yield dropped.

Example 4 Effect of Ozonation of an Aqueous Ammonia-Treated BiomassSuspension for 10 Minutes and Saccharification at Lower Enzyme Loading

To a slurry of dilute ammonia-pretreated corn cob (4.24 g of dry solid)in citrate buffer (26.6 mL, pH=5) was introduced a stream ofozone-enriched air (flow rate 2 L/min) at room temperature. After 10minutes and the consumption of 5.0 mg of ozone, the flow ofozone-enriched air was stopped and the slurry was charged withAccellerase® 1000 (393.0 μL, concentration 97.1 mg/mL) and Multifect CX12L (180.0 μL, concentration 56.1 mg/mL) enzyme cocktails, and themixture was left stirring in an incubator/shaker at 48° C. Samples weretaken every 24 h and analyzed by HPLC to determine the monomeric sugaryields versus time. Results are shown in Tables 3 and 4.

Example 5 Effect of Ozonation of an Aqueous Ammonia-Treated BiomassSuspension for 20 Minutes and Saccharification at Lower Enzyme Loading

To a slurry of dilute ammonia-pretreated corn cob (4.24 g of dry solid)in citrate buffer (26.6 mL, pH=5) was introduced a stream ofozone-enriched air (flow rate 2 L/min) at room temperature. After 20minutes and the consumption of 10.0 mg of ozone, the flow ofozone-enriched air was stopped and the slurry was charged withAccellerase® 1000 (393.0 μL, concentration 97.1 mg/mL) and Multifect CX12L (180.0 μL, concentration 56.1 mg/mL) enzyme cocktails, and themixture was left stirring in an incubator/shaker at 48° C. Samples weretaken every 24 h and analyzed by HPLC to determine the monomeric sugaryields versus time. Results are shown in Tables 3 and 4.

Example 6 Effect of Ozonation of an Aqueous Ammonia-Treated BiomassSuspension for 30 Minutes and Saccharification at Lower Enzyme Loading

To a slurry of dilute ammonia-pretreated corn cob (4.24 g of dry solid)in citrate buffer (26.6 mL, pH=5) was introduced a stream ofozone-enriched air (flow rate 2 L/min) at room temperature. After 30minutes and the consumption of 15.1 mg of ozone, the flow ofozone-enriched air was stopped and the slurry was charged withAccellerase® 1000 (393.0 μL, concentration 97.1 mg/mL) and Multifect CX12L (180.0 μL, concentration 56.1 mg/mL) enzyme cocktails, and themixture was left stirring in an incubator/shaker at 48° C. Samples weretaken every 24 h and analyzed by HPLC to determine the monomeric sugaryields versus time. Results are shown in Tables 3 and 4.

Comparative Example B Control Experiment with No Ozonation andSaccharification at Lower Enzyme Loading

To a slurry of ammonia-pretreated corn cob (4.24 g dry solid) in citratebuffer (26.6 mL, pH=5) was charged Accellerase® 1000 (393.0 μL,concentration 97.1 mg/mL) and Multifect CX 12L (180.0 μL, concentration56.1 mg/mL) enzyme cocktails, and the mixture was left stirring in anincubator/shaker at 48° C. Samples were taken every 24 h and analyzed byHPLC to determine the monomeric sugar yields versus time. Results areshown in Tables 3 and 4.

TABLE 3 Theoretical yields for glucose during saccharification ofbiomass from Examples 4-6 and Comparative Example B. SaccharificationComparative Time (hours) Example B Example 4 Example 5 Example 6 0 0 0 00 24 25.5 31.2 32.2 31.5 48 35.8 43.2 44.1 50.0 72 39.7 48.3 51.1 51.296 39.9 52.8 56.9 58.1 120 48.6 61.0 61.9 65.2 Enzyme loadings: 9 mg/gdry solid Accellerase ®, 3 mg/g dry solid Multifect.

TABLE 4 Theoretical yields for xylose during saccharification of biomassfrom Examples 5-7 and Comparative Example B. SaccharificationComparative Time (hours) Example B Example 4 Example 5 Example 6 0 0 0 00 24 19.8 22.5 24.3 24.0 48 26.6 32.0 32.8 36.4 72 29.2 35.7 37.2 36.796 29.2 38.2 40.5 40.3 120 35.3 43.8 43.3 44.7 Enzyme loadings: 9 mg/gdry solid Accellerase ®, 3 mg/g dry solid Multifect.

The results demonstrate that ozone treatment of the biomass provided areadily saccharifiable biomass and resulted in higher theoretical yieldsfor both glucose and xylose.

Ozone treatment of wet solid biomass resulted in increased yields ofboth glucose and xylose sugars.

Examples 7 through 15 illustrate a method for producing readilysaccharifiable biomass by pretreatment with a gas comprising ozonefollowed by contacting with an aqueous solution comprising ammonia. Theeffect of the pretreatment was quantified by saccharifying the readilysaccharifiable biomass to determine the theoretical yields for glucoseand xylose. Theoretical yields encompass the monomeric sugar yieldsobtained through the pretreatment and saccharification steps compared tothe total amount of sugars present in the native biomass before anypretreatment was performed. Pretreated biomass samples from Examples 7through 15 were saccharified using Accellerase® 1500 and othersaccharification enzymes.

To demonstrate the beneficial effect of the ozonation, ComparativeExamples C, D, and E were performed as control experiments following thesame procedure as Examples 7, 8, and 9 but without the ozone treatmentstep. As other control experiments, Comparative Examples F, G, and Hwere performed following the same procedure as Examples 10, 11, and 12but without the ozone treatment step. Biomass samples from ComparativeExamples C through H were saccharified using Accellerase® 1500 and othersaccharification enzymes.

For Examples 7, 8, and 9 and Comparative Examples C, D, and E,theoretical yield for glucose and xylose are given in Tables 5 and 6,respectively. For Examples 10, 11, and 12 and Comparative Examples F, G,and H, theoretical yields for glucose and xylose are given in Tables 7and 8, respectively. For Examples 13, 14, and 15, theoretical yields forglucose and xylose are given in Tables 9 and 10, respectively, alongwith results for Comparative Examples C, D, and E.

Example 7 Effect of Ozonation of Biomass Followed by Ammonia Treatmentfor 20 Minutes at 145° C.

Switchgrass (7.0 g dry material, adjusted to 60% moisture by addition ofwater) was placed in a 250 mL bottle and 5 mm Zirconium pellets (30 g)were added. The bottle was placed on a roll mill and spun at 100 rpm for90 minutes. Simultaneously, a stream of ozone-enriched air wasintroduced to the bottle; during the reaction time 75.3 mg of O₃ wasconsumed. 500 Milligrams of the ozone-treated biomass was thentransferred to a pressure vessel and mixed with aqueous ammoniumhydroxide (0.107 mL of 28% NH₃ in water), and 0.643 mL of water. Themixture was heated for 20 minutes at 145° C. Upon cooling, the resultingmaterial was dried in vacuo, and 252 mg were suspended in pH 5 buffer(1.500 mL, 14% solids loading). To the resulting slurry Accellerase®1500 (20.4 μL, 118 mg/mL) and other saccharification enzymes were added,and the mixture was left stirring in an incubator/shaker at 48° C.Samples were taken every 24 h and analyzed by HPLC to generate data onmonomeric sugar yields versus time. Results are shown in Tables 5 and 6.

Example 8 Effect of Ozonation of Biomass Followed by Ammonia Treatmentfor 60 Minutes at 145° C.

Switchgrass (7.0 g dry material, adjusted to 60% moisture by addition ofwater) was placed in a 250 mL bottle and 5 mm Zirconium pellets (30 g)were added. The bottle was placed on a roll mill and spun at 100 rpm for90 minutes. Simultaneously, a stream of ozone-enriched air wasintroduced to the bottle; during the reaction time 75.3 mg of O₃ wasconsumed. 500 Milligrams of the ozone-treated biomass was thentransferred to a pressure vessel and mixed with aqueous ammoniumhydroxide (0.107 mL of 28% NH₃ in water) and 0.643 mL of water. Themixture was heated for 60 minutes at 145° C. Upon cooling, the resultingmaterial was dried in vacuo, and 253 mg were suspended in pH 5 buffer(1.500 mL, 14% solids loading). To the resulting slurry Accellerase®1500 (20.4 μL, 118 mg/mL) and other saccharification enzymes were added,and the mixture was left stirring in an incubator/shaker at 48° C.Samples were taken every 24 h and analyzed by HPLC to generate data onmonomeric sugar yields versus time. Results are shown in Tables 5 and 6.

Example 9 Effect of Ozonation of Biomass Followed by Ammonia Treatmentfor 20 Minutes at 145° C.

Switchgrass (7.0 g dry material, adjusted to 60% moisture by addition ofwater) was placed in a 250 mL bottle and 5 mm Zirconium pellets (30 g)were added. The bottle was placed on a roll mill and spun at 100 rpm for90 minutes. Simultaneously, a stream of ozone-enriched air wasintroduced to the bottle; during the reaction time 75.3 mg O₃ wasconsumed. 500 Milligrams of the ozone-treated biomass was thentransferred to a pressure vessel and mixed with aqueous ammoniumhydroxide (0.107 mL of 28% NH₃ in water) and 0.643 mL of water. Themixture was heated for 90 minutes at 145° C. Upon cooling, the resultingmaterial was dried in vacuo, and 251 mg were suspended in pH 5 buffer(1.500 mL, 14% solids loading). To the resulting slurry Accellerase®1500 (20.4 μL, 118 mg/mL) and other saccharification enzymes were added,and the mixture was left stirring in an incubator/shaker at 48° C.Samples were taken every 24 h and analyzed by HPLC to generate data onmonomeric sugar yields versus time. Results are shown in Tables 5 and 6.

Comparative Example C Control Experiment: Ammonia Treatment for 20Minutes at 145° C. (No Ozonation)

Switchgrass (500 mg) was placed into a pressure vessel and mixed withaqueous ammonium hydroxide (0.107 mL of 28% NH₃ in water) and 0.643 mLof water. The mixture was heated for 20 minutes at 145° C. Upon cooling,the resulting material was dried in vacuo, and 250 mg were suspended inpH 5 buffer (1.500 mL, 14% solids loading). To the resulting slurryAccellerase® 1500 (20.4 μL, 118 mg/mL) and other saccharificationenzymes were added, and the mixture was left stirring in anincubator/shaker at 48° C. Samples were taken every 24 h and analyzed byHPLC to generate data on monomeric sugar yields versus time. Results areshown in Tables 5 and 6.

Comparative Example D Control Experiment: Ammonia Treatment for 60Minutes at 145° C. (No Ozonation)

Switchgrass (500 mg) was placed into a pressure vessel, and mixed withaqueous ammonium hydroxide (0.107 mL of 28% NH₃ in water) and 0.643 mLof water. The mixture was heated for 60 minutes at 145° C. Upon cooling,the resulting material was dried in vacuo, and 251 mg were suspended inpH 5 buffer (1.500 mL, 14% solids loading). To the resulting slurryAccellerase® 1500 (20.2 μL, 118 mg/mL) and other saccharificationenzymes were added, and the mixture was left stirring in anincubator/shaker at 48° C. Samples were taken every 24 h and analyzed byHPLC to generate data on monomeric sugar yields versus time. Results areshown in Tables 5 and 6.

Comparative Example E Control Experiment: Ammonia Treatment for 90Minutes at 145° C. (No Ozonation)

Switchgrass (500 mg) was placed into a pressure vessel and mixed withaqueous ammonium hydroxide (0.107 mL of 28% NH₃ in water) and 0.643 mLof water. The mixture was heated for 90 minutes at 145° C. Upon cooling,the resulting material was dried in vacuo, and 252 mg were suspended inpH 5 buffer (1.500 mL, 14% solids loading). To the resulting slurryAccellerase® 1500 (20.4 μL, 118 mg/mL) and other saccharificationenzymes were added, and the mixture was left stirring in anincubator/shaker at 48° C. Samples were taken every 24 h and analyzed byHPLC to generate data on monomeric sugar yields versus time. Results areshown in Tables 5 and 6

TABLE 5 Theoretical yields for glucose during saccharification ofbiomass from Examples 7, 8, and 9 and Comparative Examples C, D, and E.Sacch. Example Example Example Comp. Comp. Comp. Time (h) 7 8 9 Ex. CEx. D Ex. E 24 35.9 36.5 37.3 20.1 20.1 24.4 48 39.1 39.2 41.0 23.3 23.329.3 72 42.2 41.7 42.7 24.3 24.3 31.7 96 41.3 41.8 45.5 26.7 28.5 33.7120 41.7 41.0 45.8 26.4 29.5 35.0 144 41.6 41.7 46.0 26.6 29.7 35.2

TABLE 6 Theoretical yields for xylose during saccharification of biomassfrom Examples 7, 8, and 9 and Comparative Examples C, D, and E. Sacch.Example Example Example Comp. Comp. Comp. Time (h) 7 8 9 Ex. C Ex. D Ex.E 24 24.3 27.2 26.9 13.1 13.1 20.7 48 26.1 28.5 28.8 16.0 16.0 24.6 7228.2 30.4 29.9 17.2 17.2 26.6 96 27.7 30.2 31.7 19.2 21.3 28.1 120 27.929.7 32.0 19.0 21.9 29.2 144 28.0 30.2 32.1 19.5 22.2 29.2

Example 10 Effect of Ozone Treatment Followed by Ammonia Treatment

Switchgrass (7.0 g dry material, adjusted to 60% moisture by addition ofwater) was placed in a 250 mL bottle and 5 mm Zirconium pellets (30 g)were added. The bottle was placed on a roll mill and spun at 100 rpm for90 minutes. Simultaneously, a stream of ozone-enriched air wasintroduced to the bottle; during the reaction time 75.3 mg O₃ wasconsumed. 500 Milligrams of the ozone-treated biomass was thentransferred to a pressure vessel and mixed with aqueous ammoniumhydroxide (0.107 mL of 28% NH₃ in water) and 0.643 mL of water. Themixture was heated for 20 minutes at 155° C. Upon cooling, the resultingmaterial was dried in vacuo, and 246 mg were suspended in pH 5 buffer(1.500 mL, 14% solids loading). To the resulting slurry Accellerase®1500 (19.9 μL, 118 mg/mL) and other saccharification enzymes were added,and the mixture was left stirring in an incubator/shaker at 48° C.Samples were taken every 24 h and analyzed by HPLC to generate data onmonomeric sugar yields versus time. Results are shown in Tables 7 and 8.

Example 11 Effect of Ozonation of Biomass Followed by Ammonia Treatmentfor 60 Minutes at 155° C.

Switchgrass (7.0 g dry material, adjusted to 60% moisture by addition ofwater) was placed in a 250 mL bottle and 5 mm Zirconium pellets (30 g)were added. The bottle was placed on a roll mill and spun at 100 rpm.Simultaneously, a stream of ozone-enriched air was introduced to thebottle; during the reaction time 75.3 mg O₃ was consumed. 500 Milligramsof this material was then transferred to a pressure vessel, and mixedwith aqueous ammonium hydroxide (0.107 mL of 28% NH₃ in water) and 0.643mL of water. The mixture was heated for 60 minutes at 155° C. Uponcooling, the resulting material was dried in vacuo, and 257 mg weresuspended in pH 5 buffer (1.500 mL, 14% solids loading). To theresulting slurry Accellerase® 1500 (20.8 μL, 118 mg/mL) and othersaccharification enzymes were added, and the mixture was left stirringin an incubator/shaker at 48° C. Samples were taken every 24 h andanalyzed by HPLC to generate data on monomeric sugar yields versus time.Results are shown in Tables 7 and 8.

Example 12 Effect of Ozonation of Biomass Followed by Ammonia Treatmentfor 90 Minutes at 155° C.

Switchgrass (7.0 g dry material, adjusted to 60% moisture by addition ofwater) was placed in a 250 mL bottle and 5 mm Zirconium pellets (30 g)were added. The bottle was placed on a roll mill and spun at 100 rpm for90 minutes. Simultaneously, a stream of ozone-enriched air wasintroduced to the bottle; during the reaction time 75.3 mg O₃ wasconsumed. 500 Milligrams of this material was then transferred to apressure vessel, and mixed with aqueous ammonium hydroxide (0.107 mL of28% NH₃ in water) and 0.643 mL of water. The mixture was heated for 90minutes at 155° C. Upon cooling, the resulting material was dried invacuo, and 248 mg were suspended in pH 5 buffer (1.500 mL, 14% solidsloading). To the resulting slurry Accellerase® 1500 (20.1 μL, 118 mg/mL)and other saccharification enzymes were added, and the mixture was leftstirring in an incubator/shaker at 48° C. Samples were taken every 24 hand analyzed by HPLC to generate data on monomeric sugar yields versustime. Results are shown in Tables 7 and 8.

Comparative Example F Control Experiment: Ammonia Treatment for 20Minutes at 155° C. (No Ozonation)

Switchgrass (500 mg dry, 533 mg wet) was placed into a pressure vesseland mixed with aqueous ammonium hydroxide (0.107 mL of 28% NH₃ in water)and 0.643 mL of water. The mixture was heated for 20 minutes at 155° C.Upon cooling, the resulting material was dried in vacuo, and 256 mg weresuspended in pH 5 buffer (1.500 mL, 14% solids loading). To theresulting slurry Accellerase® 1500 (20.7 μL, 118 mg/mL) and othersaccharification enzymes were added, and the mixture was left stirringin an incubator/shaker at 48° C. Samples were taken every 24 h andanalyzed by HPLC to generate data on monomeric sugar yields versus time.Results are shown in Tables 7 and 8.

Comparative Example G Control Experiment: Ammonia Treatment for 60Minutes at 155° C. (No Ozonation)

Switchgrass (500 mg dry, 533 mg wet) was placed into a pressure vessel,and mixed with aqueous ammonium hydroxide (0.107 mL of 28% NH₃ in water)and 0.643 mL of water. The mixture was heated for 60 minutes at 155° C.Upon cooling, the resulting material was dried in vacuo, and 249 mg weresuspended in pH 5 buffer (1.500 mL, 14% solids loading). To theresulting slurry Accellerase® 1500 (20.1 μL, 118 mg/mL) and othersaccharification enzymes were added, and the mixture was left stirringin an incubator/shaker at 48° C. Samples were taken every 24 h andanalyzed by HPLC to generate data on monomeric sugar yields versus time.Results are shown in Tables 7 and 8.

Comparative Example H Control Experiment: Ammonia Treatment for 90Minutes at 155° C. (No Ozonation)

Switchgrass (500 mg dry, 533 mg wet) was placed into a pressure vessel,and mixed with aqueous ammonium hydroxide (0.107 mL of 28% NH₃ in water)and 0.643 mL of water. The mixture was heated for 90 minutes at 155° C.Upon cooling, the resulting material was dried in vacuo, and 253 mg weresuspended in pH 5 buffer (1.500 mL, 14% solids loading). To theresulting slurry Accellerase® 1500 (20.5 μL, 118 mg/mL) and othersaccharification enzymes were added, and the mixture was left stirringin an incubator/shaker at 48° C. Samples were taken every 24 h andanalyzed by HPLC to generate data on monomeric sugar yields versus time.Results are shown in Tables 7 and 8.

TABLE 7 Theoretical yields for glucose during saccharification ofbiomass from Examples 10, 11, and 12 and Comparative Examples F, Sacch.Example Example Example Comp. Comp. Comp. Time (h) 10 11 12 Ex. F Ex. GEx. H 24 36.9 37.4 39.5 24.1 29.1 29.1 48 38.5 40.1 43.7 27.2 33.6 33.872 39.9 42.2 44.6 28.8 37.7 37.5 96 42.5 44.8 45.7 31.2 39.4 38.0 12038.5 42.8 43.9 29.7 39.1 39.1 144 41.7 44.2 44.9 31.6 40.6 42.1

TABLE 8 Theoretical yields for xylose during saccharification of biomassfrom Examples 10, 11, and 12 and Comparative Examples F, G, and H.Sacch. Example Example Example Comp. Comp. Comp. Time (h) 10 11 12 Ex. FEx. G Ex. H 24 26.5 26.5 28.4 18.3 30.2 29.9 48 26.8 28.2 30.2 21.1 32.932.7 72 27.4 29.2 30.6 22.3 35.8 35.5 96 29.2 30.6 31.0 24.2 36.6 34.9120 27.2 29.9 30.3 23.6 36.7 36.2 144 29.4 31.0 30.7 25.1 37.8 38.7

The data show that prolonged ozonolysis and prolonged ammoniapretreatment result in xylose degradation. Hemicellulose, a lessrecalcitrant component than cellulose, is likely degraded under harsherpretreatment conditions.

Examples 13 through 15 describe pretreatments using 120 minutes ofozonation, and 145° C. ammonia pretreatment for 20, 60, and 90 minutes.Comparative Examples C, D, and E represent their respective controls.

Example 13 Effect of Ozonation of Biomass Followed by Ammonia Treatmentfor 20 Minutes at 145° C.

Switchgrass (7.0 g dry material, adjusted to 60% moisture by addition ofwater) was placed in a 250 mL bottle and 5 mm Zirconium pellets (30 g)were added. The bottle was placed on a roll mill and spun at 100 rpm for120 minutes. Simultaneously, a stream of ozone-enriched air wasintroduced to the bottle; during the reaction time 100.4 mg of O₃ wasconsumed. 500 Milligrams of the ozone-treated biomass was thentransferred to a pressure vessel and mixed with aqueous ammoniumhydroxide (0.107 mL of 28% NH₃ in water), and 0.643 mL of water. Themixture was heated for 20 minutes at 145° C. Upon cooling, the resultingmaterial was dried in vacuo, and 251 mg were suspended in pH 5 buffer(1.491 mL, 14% solids loading). To the resulting slurry Accellerase®1500 (20.3 μL, 118 mg/mL) and other saccharification enzymes were added,and the mixture was left stirring in an incubator/shaker at 48° C.Samples were taken every 24 h and analyzed by HPLC to generate data onmonomeric sugar yields versus time. Results are shown in Tables 9 and10.

Example 14 Effect of Ozonation of Biomass Followed by Ammonia Treatmentfor 60 Minutes at 145° C.

Switchgrass (7.0 g dry material, adjusted to 60% moisture by addition ofwater) was placed in a 250 mL bottle and 5 mm Zirconium pellets (30 g)were added. The bottle was placed on a roll mill and spun at 100 rpm for120 minutes. Simultaneously, a stream of ozone-enriched air wasintroduced to the bottle; during the reaction time 100.0 mg of O₃ wasconsumed. 500 Milligrams of the ozone-treated biomass was thentransferred to a pressure vessel and mixed with aqueous ammoniumhydroxide (0.107 mL of 28% NH₃ in water) and 0.643 mL of water. Themixture was heated for 60 minutes at 145° C. Upon cooling, the resultingmaterial was dried in vacuo, and 247 mg were suspended in pH 5 buffer(1.471 mL, 14% solids loading). To the resulting slurry Accellerase®1500 (20.0 μL, 118 mg/mL) and other saccharification enzymes were added,and the mixture was left stirring in an incubator/shaker at 48° C.Samples were taken every 24 h and analyzed by HPLC to generate data onmonomeric sugar yields versus time. Results are shown in Tables 9 and10.

Example 15 Effect of Ozonation of Biomass Followed by Ammonia Treatmentfor 20 Minutes at 145° C.

Switchgrass (7.0 g dry material, adjusted to 60% moisture by addition ofwater) was placed in a 250 mL bottle and 5 mm Zirconium pellets (30 g)were added. The bottle was placed on a roll mill and spun at 100 rpm for120 minutes. Simultaneously, a stream of ozone-enriched air wasintroduced to the bottle; during the reaction time 75.3 mg O₃ wasconsumed. 500 Milligrams of the ozone-treated biomass was thentransferred to a pressure vessel and mixed with aqueous ammoniumhydroxide (0.107 mL of 28% NH₃ in water) and 0.643 mL of water. Themixture was heated for 90 minutes at 145° C. Upon cooling, the resultingmaterial was dried in vacuo, and 247 mg were suspended in pH 5 buffer(1.471 mL, 14% solids loading). To the resulting slurry Accellerase®1500 (20.0 μL, 118 mg/mL) and other saccharification enzymes were added,and the mixture was left stirring in an incubator/shaker at 48° C.Samples were taken every 24 h and analyzed by HPLC to generate data onmonomeric sugar yields versus time. Results are shown in Tables 9 and10.

TABLE 9 Theoretical yields for glucose during saccharification ofbiomass from Examples 13, 14, and 15 and Comparative Examples C, D, andE. Sacch. Example Example Example Comp. Comp. Comp. Time (h) 13 14 15Ex. C Ex. D Ex. E 24 39.8 39.7 39.8 20.1 20.1 24.4 48 44.7 45.3 44.823.3 23.3 29.3 72 43.0 43.1 44.1 24.3 24.3 31.7 96 45.6 45.6 45.5 26.728.5 33.7 120 47.3 47.9 47.9 26.4 29.5 35.0

TABLE 10 Theoretical yields for xylose during saccharification ofbiomass from Examples 13, 14, and 15 and Comparative Examples C, D, andE. Sacch. Example Example Example Comp. Comp. Comp. Time (h) 13 14 15Ex. C Ex. D Ex. E 24 28.5 30.3 31.7 13.1 13.1 20.7 48 30.6 32.5 33.016.0 16.0 24.6 72 30.0 31.4 32.8 17.2 17.2 26.6 96 33.1 33.6 33.7 19.221.3 28.1 120 35.0 34.7 34.7 19.0 21.9 29.2

Although particular embodiments of the present invention have beendescribed in the foregoing description, it will be understood by thoseskilled in the art that the invention is capable of numerousmodifications, substitutions, and rearrangements without departing fromthe spirit of essential attributes of the invention. Reference should bemade to the appended claims, rather than to the foregoing specification,as indicating the scope of the invention.

1. A method for producing readily saccharifiable carbohydrate-enrichedbiomass, the method comprising: (a) providing lignocellulosic biomasscomprising lignin; (b) contacting the biomass with an aqueous solutioncomprising ammonia to form a biomass-aqueous ammonia mixture, whereinthe ammonia is present at a concentration at least sufficient tomaintain alkaline pH of the biomass-aqueous ammonia mixture but whereinsaid ammonia is present at less than about 12 weight percent relative todry weight of biomass, and further wherein the dry weight of biomass isat a high solids concentration of at least about 15 weight percentrelative to the weight of the biomass-aqueous ammonia mixture, toproduce an ammonia-treated biomass; and (c) contacting theammonia-treated biomass with a gas comprising ozone at a temperature ofabout 0° C. to about 50° C., whereby a readily saccharifiablecarbohydrate-enriched biomass is produced.
 2. A method for producingreadily saccharifiable carbohydrate-enriched biomass, the methodcomprising: (a) providing lignocellulosic biomass comprising lignin; (b)contacting the biomass with a gas comprising ozone at a temperature ofabout 0° C. to about 50° C. (c) contacting the ozone-treated biomasswith an aqueous solution comprising ammonia to form a mixture comprisingozone-treated biomass and aqueous ammonia, wherein the ammonia ispresent at a concentration at least sufficient to maintain alkaline pHof the mixture but wherein said ammonia is present at less than about 12weight percent relative to dry weight of ozone-treated biomass, andfurther wherein the dry weight of biomass is at a high solidsconcentration of at least about 15 weight percent relative to the weightof the mixture, whereby a readily saccharifiable carbohydrate-enrichedbiomass is produced.
 3. The method of claim 1 or 2, wherein the gascomprises about 0.1 to about 20 percent by volume ozone.
 4. The methodof claim 1 or 2, wherein the gas further comprises air, nitrogen,oxygen, argon, or a combination thereof.
 5. The method of claim 1,wherein the ratio of ozone to ammonia-treated biomass in step (c) is atleast 1:100 on a weight basis.
 6. The method of claim 2, wherein theratio of ozone to lignocellulosic biomass in step (b) is at least 1:100on a weight basis.
 7. The method of claim 1, further comprising applyingenergy to the lignocellulosic biomass during step (b), to theammonia-treated biomass during step (c), or to both.
 8. The method ofclaim 2, further comprising applying energy to the lignocellulosicbiomass during step (b), to the ozone-treated biomass during step (c),or to both.
 9. The method of claim 1 or 2, wherein the lignocellulosicbiomass, the ammonia-treated biomass, or both contain at least about 30percent moisture.
 10. The method of claim 1 or 2, wherein ammonia isselected from the group consisting of ammonia gas, ammonium hydroxide,urea, and combinations thereof.
 11. The method of claim 1 or 2, whereinthe aqueous solution comprising ammonia further comprises at least oneadditional base selected from the group consisting of sodium hydroxide,sodium carbonate, potassium hydroxide, potassium carbonate, calciumhydroxide, and calcium carbonate.
 12. The method of claim 1 or 2,further comprising saccharifying the readily saccharifiablecarbohydrate-enriched biomass with an enzyme consortium wherebyfermentable sugars are produced.
 13. The method of claim 12, furthercomprising fermenting the sugars to produce a target product.